Peptidoglycan is a major component of the bacterial cell wall that gives the wall its shape and strength. It is unique to bacteria and found in all bacteria, both gram-positive and gram-negative. Peptidoglycan is a polymer of glycan strands that are cross-linked through short peptide bridges. It consists of alternating β1-4 linked residues of N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc). A pentapeptide chain is attached to MurNAc (MurNAc-pentapeptide) and cross-linking occurs between these peptide chains.
Biosynthesis of peptidoglycan can be divided into three stages: firstly, synthesis of the precursors in the cytoplasm, secondly, transfer of the precursors to a lipid carrier molecule and, thirdly, insertion of the precursors into the cell wall and coupling to existing peptidoglycan.
The precursors synthesised in the cytoplasm are the sugar nucleotides: UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide).
The second stage, which occurs in the cytoplasmic membrane, is catalysed by two enzymes and involves synthesis of a disaccharide unit on a lipid carrier, undecaprenyl phosphate. The lipid carrier is also involved in the synthesis of other components of the bacterial cell wall.
The first enzyme catalyses the transfer of phosphoryl-N-acetyl muramyl pentapeptide from UDP-MurNAc-pentapeptide to undecaprenol phosphate with the simultaneous release of UMP. This enzyme is called phospho-N-acetylmuramyl-pentapeptide translocase (hereafter referred to as “the translocase”) and is the product of the gene mraY in Escherichia coli. The product, undecaprenol-pyrophosphate-N-acetylmuramylpentapeptide (Lipid-P-P-MurNAc-pentapeptide) or Lipid I or Lipid linked precursor I is the substrate for the second enzyme.
N-acetylglucosaminyl transferase, transfers N-acetylglucosamine from UDP-GlcNAc (with simultaneous release of UDP) to form undecaprenol-pyrophosphoryl-N-acetylmuramylpentapeptide-N-acetylglucosamine or Lipid II or Lipid linked precursor II. This enzyme is also called UDP-N-acetylglucosamine: N-acetylmuramyl(pentapeptide)-P-P-undecaprenol-N-acetylglucosamine transferase (hereafter referred to as “the transferase”). The enzyme is the product of the gene murG in Escherichia coli. 
The translocase and the transferase enzymes are essential for bacterial viability (see respectively D. S. Boyle and W. D. Donachie, J. Bacteriol. (1998), 180, 6429–6432 and D. Mengin-Lecreulx, L. Texier, M. Rousseaue and J. Van Heijernoot, J. Bacteriol. (1991), 173, 4625–4636).
In the third stage, at the exterior of the cytoplasmic membrane, polymerisation of the glycan occurs. The disaccharide-pentapeptide unit is transferred from the lipid carrier to an existing disaccharide unit or polymer by a peptidogycan transglycosylase (also referred to as a peptidoglycan polymerase) (hereafter referred to as “the transglycosylase”). The joining of the peptide bridge is catalyzed by peptidoglycan transpeptidase (hereafter referred to as “the transpeptidase”). Both enzyme activities, which are essential, reside in the same molecule, the penicillin-binding proteins (or PBPs), as in PBP 1a or 1b in Escherichia coli. These are the products of the ponA and ponB genes respectively, in Escherichia coli. 
On transfer of the disaccharide-pentapeptide unit from the lipid precursor to an existing peptidoglycan chain the lipid is released as a molecule of undecaprenol pyrophosphate. This has to be cleaved by a bacitracin-sensitive undecaprenyl pyrophosphorylase, also called undecaprenol pyrophosphorylase or C55-isoprenyl pyrophosphorylase (hereafter referred to as the “lipid pyrophosphorylase”) to generate undecaprenol phosphate which can then re-enter the cycle at the second stage. Since inhibition of this enzyme will inhibit recycling of the lipid precursor it could also inhibit formation of peptidoglycan.
The transglycosylase is usually assayed by radiolabelling one of the sugar molecules and monitoring its incorporation into peptidoglycan. It is a difficult enzyme to assay because the lipid carrier molecule with bound disaccharide is neither simple to make nor water-soluble and, furthermore, the reaction only occurs on a solid phase (e.g. on Whatman 3 mm paper) and so the reaction conditions are difficult to control.
The transglycosylase activity may alternatively be assayed indirectly in a solution phase assay which, whilst being easier to control, requires the use of three of the other key enzymes involved in peptidoglycan synthesis, the translocase (e.g. the mraY gene product), the transferase (e.g. the murG gene product) and the lipid pyrophosphorylase.
In both types of assay, quantification of the products of enzymatic reaction is carried out using paper chromatography in which peptidoglycan stays at the origin and the reactants move away from the origin.
It would be desirable to develop an assay for detecting peptidoglycan synthesis which dispensed with the need for paper chromatography altogether. More particularly, it would be desirable to develop an assay for detecting peptidoglycan synthesis in which the reaction and quantification of the products of reaction could be performed entirely in the solution phase, for example, in a microtitre plate.